Hemoglobin measurement from a single vessel

ABSTRACT

A system and method to measure blood oxygenation levels and total hemoglobin on individually selected blood vessels, to provide a representation of the subject condition and of tissue perfusion that may be used for diagnosing specific tissue conditions. Reflection spectra from individual blood vessels or a collection of vessels are measured by using wide-field imaging for selecting target vessels and a narrow-field confocal detection system to enable measuring local blood oxygenation and hemoglobin. Optical fibers may be used to illuminate the target vessel and to detect light diffusively reflected therefrom. The reflection spectra may be analyzed in a spectrometer to extract the ratio of the deoxy- to oxyhemoglobin and to determine their absolute concentration for computing total hemoglobin levels. An alternative implementation uses image processing on camera images of a blood vessel, generated at an isosbestic wavelength of the deoxy- and oxyhemoglobin, and optionally also at neighboring wavelengths.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/779,604, filed on May 29, 2018, which is a National Phase of PCTPatent Application No. PCT/IL2016/051286 having International filingdate of Nov. 30, 2016, which claims the benefit of priority of U.S.Provisional Patent Application No. 62/260,978, filed on Nov. 30, 2015.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of pulse oximetry, especially thatproviding information on hemoglobin levels in individual blood vessels.

BACKGROUND

Hemoglobin is the metalloprotein that carries the oxygen in the redblood cells, and makes up about 97% of the dry content of these cells. Asingle Hemoglobin molecule can bind up to four oxygen molecules presentin the lungs for releasing in the tissues.

Several methods exist for measuring blood oxygen saturation. Thearterial blood gas (ABG) test uses blood collected directly from anartery into a vacuum tube that maintains the oxygenation level. The gascontent in the collected blood sample is then tested to measure arterialoxygen tension (PaO2). Pulse oximetry measures the arterial oxygensaturation optically by exploiting the different absorption spectra ofhemoglobin and oxyhemoglobin, indicated in FIG. 1, as well as thepulsatile nature of the arterial flow. An advantage of pulse oximetry isits noninvasiveness, which both reduces risk of infection and othercomplications arising from invasive techniques, and eliminates pain.

Typically, pulse oximetry measures the absorption of two or more opticalwavelengths by the red blood cells. Light at two wavelength bands may betransmitted through the patient's finger, or ear lobe, illuminatingtissue lying therebelow, such as blood vessels, which scatter the light.The scattered light is measured as it leaves the tissue to indicateoxygenation levels. The first band, typically selected at approximately800 nm, where oxy- and deoxy-hemoglobin have approximately equalabsorption, serves as a reference to the total amount of hemoglobin inthe optical path. The second band, typically in the region of 950 nm,may be absorbed very differently by the two hemoglobin forms, and may beused to indicate the oxygen saturation level within the blood.

While measuring the level of oxygenation is important in arterial bloodfor assessing heart and lung function, it may also be used to measureblood oxygenation in capillaries, which are the primary location for theexchange of oxygen and carbon dioxide between the blood and the tissue.For example, such measurement could provide information of tissueconditions and hypoxia, and serve as a tool to measure local tissueviability during invasive surgery. In addition, knowledge of bloodoxygenation in veins is an important clinical parameter; however, itdoes not provide direct information on oxygen perfusion into the tissue,as large portion of the blood are transferred directly from the arteriesto the veins without passing through the capillary network. Thus, venousblood oxygenation is not always an accurate indication on tissueperfusion.

Conventional pulse oximetry performs measurements on light diffusedwithin the tissue in order to assess the arterial blood oxygenationlevels. Thus, the optical measurement obtained by these methods oftencombines the absorption of many blood vessels, such as large arteries,arterioles, arterial capillaries, venous capillaries, venules, and largeveins. To separate the different blood oxygenation levels of thedifferent types of vessels, pulse oximetry often relies on the temporalpulsation of the arterial blood volume, allowing the subtraction of theconstant background light attenuation. Hence, pulse oximetry is limitedin that it measures blood oxygenation levels in pulsed vessels only,i.e. arterial vessels only, such that there exists a need for ameasurement system that enables the blood oxygenation levels to bemeasured also in venous vessels, or in groups of vessels having similarphysiological properties but not limited to any specific type.

Prior art optical methods mainly concentrate on determination of theoxygenation levels in blood vessels tested based on the measured ratioof oxyhemoglobin to deoxyhemoglobin. Such measurements are described inGerman Patent Application published as DE 4433827 A1, by FriedrichSchiller University, Jena, for (freely translated) “Method and apparatusfor measuring substance parameters in material layers, especiallycalibration-free, in-vivo, oxygen saturation in optically accessibleblood containing structures”, and in International Patent Applicationpublished as WO 2006/129740 by Olympus Corporation et al., for“Hemoglobin observing device and hemoglobin observing method”.

However, the total hemoglobin level is also an important parameternecessary for assessing the patient's general health status. The mainchallenge in measuring hemoglobin concentration is the need to measurean absolute absorption parameter, in contrast to the above mentionedblood oxygenation parameters, that require measurement of only the ratiobetween absorption spectra. There therefore exists a need for simple,non-invasive optical methods and apparatus for making in-vivomeasurements of the total hemoglobin level in blood vessels.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the figures.

SUMMARY

A system and method are disclosed herein to measure blood oxygenationand total hemoglobin on individually selected blood vessels, to providea representation of tissue perfusion that may be used for diagnosingspecific tissue conditions. Reflection spectra from individual bloodvessels can be measured by using wide-field illumination and anarrow-field confocal detection system to enable high resolutionselection and focus onto a target vessel for measuring blood oxygenationand hemoglobin. Optical fibers may be used to illuminate the targetvessel and to detect light reflected therefrom.

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope.

There is thus provided in accordance with an exemplary implementationdescribed in this disclosure, a method for measuring total hemoglobinconcentration in a blood vessel, comprising:

(i) illuminating a bodily tissue containing the blood vessel,

(ii) generating images of the blood vessel and its surrounding tissue ata first wavelength in the region of an isosbestic wavelength ofoxyhemoglobin and deoxy-hemoglobin,

(iii) determining at the first wavelength, the comparative imagedintensities of the blood vessel and of the surrounding tissue,

(v) estimating the optical path length of the illumination through thevessel, and

(vi) using the estimated optical path length and the comparative imagedintensities of the blood vessel and of the surrounding tissue todetermine the total hemoglobin concentration in the blood flowing in theblood vessel. In such a method, the step of estimating the optical pathlength of the illumination through the vessel may be obtained afterdetermining the size of the vessel from at least one of the generatedimages.

The above described method may further comprise:

(vii) generating images of the blood vessel and its surrounding tissueat at least one additional wavelength in proximity to the firstwavelength,

(viii) determining the comparative imaged intensities of the bloodvessel at the first wavelength and at the at least one additionalwavelength in proximity thereto, and

(ix) using the comparative imaged intensities of the blood vessel todetermine the oxygen absorption level in the blood. In such a case, theat least one additional wavelength may be a range of multiplewavelengths.

In any of the above described methods, the images of the blood vesseland its surrounding tissue may be obtained from a camera system, or froma narrow field confocal microscope system. Additionally, the firstwavelength images may be generated by illuminating at the firstwavelength, or by imaging at the first wavelength.

According to yet further implementations of the present disclosure,there is further provided a system for measuring total hemoglobinconcentration in a blood vessel, comprising:

(i) a light source configured to illuminate the blood vessel and itssurrounding tissue,

(ii) a camera configured to generate images of the blood vessel and itssurrounding tissue at a first wavelength in the region of an isosbesticwavelength of oxyhemoglobin and deoxy-hemoglobin, and

(iii) a processor configured to:

-   -   (a) determine from at least one of the images generated at the        first wavelength, the comparative imaged intensities of the        blood vessel and of the surrounding tissue,    -   (b) estimate the optical path length of the illumination through        the blood vessel, and    -   (c) use the estimated optical path length and the comparative        imaged intensities of the blood vessel and of the surrounding        tissue to determine the total hemoglobin concentration in the        blood flowing in the blood vessel. The estimating of the optical        path length of the illumination through the vessel may be        obtained after determining the size of the vessel from at least        one of the generated images.

According to a further implementation of the described system, thecamera may be configured to generate images of the blood vessel and itssurrounding tissue at at least one additional wavelength in proximity tothe first wavelength, and in addition, the processor may be furtherconfigured to

-   -   (d) determine the comparative imaged intensities of the blood        vessel at the first wavelength and at the at least one        additional wavelength in proximity thereto, and    -   (e) use the comparative imaged intensities of the blood vessel        to determine the oxygen absorption level in the blood. In such a        system the at least one additional wavelength may be a range of        multiple wavelengths.

In any of the above described systems the light source may be configuredto provide wide field illumination. Furthermore, the camera may comprisea confocal microscope system configured to image the blood vessel andits surrounding tissue.

According to additional configurations of the above described systems,the first wavelength images may be generated by illuminating at thefirst wavelength, or by use of a light source providing illumination atthe first wavelength. The first wavelength images may be generated by atleast one filter disposed in the optical path of the illumination eitherbefore incidence on the blood vessel and its surrounding tissue, orafter reflection from the blood vessel and its surrounding tissue.

There are described in the present disclosure, yet furtherimplementations of apparatus, for determining the oxygen level andhemoglobin in at least one blood vessel of a subject, comprising:

(i) a light source configured to illuminate a bodily tissue with aplurality of wavelengths,

(ii) a light collector configured to collect light diffusively scatteredfrom the bodily tissue,

(iii) a spectrum detector configured to receive the collected light anddetermine a spectrum corresponding to the collected light, and

(iv) a processor configured to determine a blood oxygen absorption levelcorresponding to the determined spectrum,

wherein the light source and the light collector may comprise a confocaldetection system having a detection volume such that the detectorreceives light only from a region containing the at least one vessel ofthe subject.

In such an apparatus, the at least one blood vessel may be either asingle blood vessel or a group of vessels of similar physiologicalproperties. Additionally, the spectrum detector may be a spectrometer.

According to further implementations, the apparatus may further comprisea camera in optical communication with the light collector, andconfigured to image the bodily tissue using the collected light, therebyallowing the light source and the light collector to be maneuvered ontoany of a single selected blood vessel and tissue surrounding the atleast one blood vessel. In such a case, the processor should beconfigured to determine the oxygen absorption level of the at least oneblood vessel by analyzing i) a reference spectrum corresponding to thetissue surrounding the at least one blood vessel, wherein the referencespectrum does not include spectral information corresponding to the atleast one blood vessel, and ii) a blood vessel spectrum correspondingonly to the at least one blood vessel.

The apparatus then may further comprise:

(i) a first fiber coupled to the light source and having a relativelylarge core, and configured to illuminate the bodily tissue with awidefield beam having an illumination region comparable to thecross-section of the at least one blood vessel,

(ii) a detection channel providing the optical communication between thelight collector and the camera, thereby providing the camera withcollected light having a collection volume corresponding to thewidefield beam, and

(iii) a second fiber in optical communication with the light collectorand coupled to the spectrum detector, wherein the second fiber has coresize providing the spectrum detector with collected light having acollection volume that is of the order of the cross-section of the atleast one blood vessel. In any of the above described apparatus, the atleast one blood vessel may be at least one capillary.

Finally, as an alternative to the above described apparatusimplementations, there is further provided an apparatus for determiningthe oxygen absorption level in a blood vessel of a subject, comprising:

(i) a light source configured to illuminate a bodily tissue withwavelengths at an isosbestic wavelength of oxyhemoglobin anddeoxy-hemoglobin, and at wavelengths in proximity thereto,

(ii) a camera configured to generate images of the blood vessel and itssurrounding tissue at the isosbestic wavelength and at the wavelengthsin proximity thereto, and

(iii) an image processor configured to

-   -   (a) determine the comparative imaged intensities of the blood        vessel at the isosbestic wavelength and at the wavelengths in        proximity thereto, and    -   (b) determine the oxygen absorption level in the blood vessel        from the comparative imaged intensities of the blood vessels        determined in step (i).

According to yet further implementations of the present disclosure,there is provided a method for determining the oxygen level andhemoglobin in at least one blood vessel of a subject, comprising:

(i) illuminating a bodily tissue with a plurality of wavelengths,

(ii) collecting light diffusively scattered from the bodily tissue,

(iii) determining a spectrum corresponding to the received light, and

(iv) determining a blood oxygen absorption level corresponding to thedetermined spectrum,

wherein the light source and the light collector may comprise a confocaldetection system having a detection volume such that the light collectorreceives light only from a region containing the at least one bloodvessel of the subject. In such a method, the at least one blood vesselmay be either a single blood vessel or a group of vessels of similarphysiological properties. The single blood vessel may be a capillary.

Additional implementations of the above method may invoke:

(i) imaging the bodily tissue using the collected light, and

(ii) directing the light source and the light collector onto any of asingle selected blood vessel and tissue surrounding the selected singleblood vessel using the images. In either of the previous two methods,the step of determining the blood oxygen absorption level correspondingto the determined spectrum may comprise determining the blood oxygenabsorption level of the selected single blood vessel by analyzing i) areference spectrum corresponding to the tissue surrounding the singleblood vessel, wherein the reference spectrum does not include spectralinformation corresponding to the single selected blood vessel, and ii) ablood vessel spectrum corresponding only to the selected single bloodvessel. In such a case, the method may further comprise:

(i) illuminating the bodily tissue with a widefield beam having a pointspread function comparable to the width of the selected single bloodvessel,

(ii) imaging the bodily tissue with collected light having a collectionvolume corresponding to the widefield beam, thereby maneuvering thelight source and light collector in accordance with a resolutionassociated with the widefield beam, and

(iii) providing a spectrum detector with the portion of the collectedlight having the collection volume that is smaller than thecross-section of the selected single blood vessel, thereby determiningthe blood oxygen absorption level of the selected single blood vessel.

Finally, as an alternative to the above described methods, there isfurther provided a method for determining the blood oxygen level andhemoglobin in a blood vessel of a subject comprising:

(i) illuminating a bodily tissue with wavelengths at an isosbesticwavelength of oxyhemoglobin and deoxy-hemoglobin, and at wavelengths inproximity thereto,

(ii) generating images of the blood vessel and its surrounding tissue atthe isosbestic wavelength and at the wavelengths in proximity thereto,

(iii) determining the comparative imaged intensities of the blood vesselat the isosbestic wavelength and at the wavelengths in proximitythereto, and

(iv) using the comparative imaged intensities of the blood vessel todetermine the oxygen absorption level in the blood.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thefigures and by study of the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. Dimensionsof components and features shown in the figures are generally chosen forconvenience and clarity of presentation and are not necessarily shown toscale. The figures are listed below.

FIG. 1 shows absorption spectra for hemoglobin and oxyhemoglobin;

FIG. 2A shows an exemplary implementation of an optical apparatus formeasuring reflection spectra from individual blood vessels;

FIG. 2B shows an example of a zoomed in view of a blood vessel crosssection illuminated and measured with the apparatus of FIG. 2A;

FIG. 2C shows multiple different types of blood vessels beneath thetissue surface illuminated separately to allow collecting individualspectra;

FIGS. 3A-C, show typical images of capillaries and surrounding tissue onthe lip of a subject;

FIG. 4 shows a flowchart of an exemplary method for measuring bloodoxygenation levels using the system of FIG. 2A; and

FIGS. 5A-G show a method to determine oxygenation of a blood vessel, inaccordance with another implementation of the present disclosure.

DETAILED DESCRIPTION

Reference is first made to FIG. 1, which shows the absorption spectrafor hemoglobin and oxyhemoglobin from the visible through the nearinfra-red region, showing the characteristic isosbestic points, one inthe infra-red at approximately 805 nm, and others in the visible in the500 to 600 nm region, such as 569 and 586 nm.

Reference is now made to FIG. 2A, which illustrates an exemplary opticalapparatus 100 for measuring reflection spectra from individual bloodvessels, in accordance with one implementation of the systems describedin this disclosure. A light source 102 coupled to a multimode fiber 104may emit broadband light which is channeled through fiber 104 andcollimated by a lens 106. The collimated light may be projected via anobjective lens 108 onto a tissue surface 110 covering a blood vessel112.

Optical apparatus 100 generates a confined illumination distributionover blood vessel 112. For example, one or more parameters of opticalapparatus 100 may be selected to produce an illumination beam havingdimensions in the order of the width of vessel 112, or slightly larger.Alternatively, the beam illuminates a larger region that contains aplurality of vessels having similar physiological characteristic, forexample arterioles, or venules or capillaries. Fiber 104 may be selectedto have a relatively large core, such as a multimode fiber having a corein the order of 50 micrometers (μm) and the parameters of lenses 106,and 108 may be selected accordingly, to produce the desired illuminationbeam dimensions.

Objective lens 108 may collect light scattered by illuminated tissue110. A beam splitter 116 directs the collected light to a spectrometer114 via a coupling lens 118 and a single-mode fiber 120. Fiber core 120may be selected to have a relatively small diameter, such as in theorder of 5 μm, resulting in a light collection region that is smallerthan the illumination region. The resulting point-spread function (PSF)of the combined illumination and collection optical paths may havedimensions that are comparable to the cross-section of the target vesselor group of vessels 112, and as a result, may allow obtaining a spectralmeasurement from the vessels 112 without including spectral informationfrom nearby and/or deeper vessels. The apparatus 100 may mostconveniently be constructed as a single readily maneuverable unit.Alternatively, the optical source 102, the spectrometer 114, the camera130, the processor 134, and any display unit (not shown) may beincorporated into a separate static module, connected to a moveablescanning optical head by means of the fiber optical and electroniclinks, or any other suitable arrangement may be used.

Referring to FIG. 2B, a zoomed in view of vessel 112 is shown. Therepresentation 122 of the illuminated region produced by opticalapparatus 100 should have a width that is comparable to or slightlylarger than the width of vessel 112, whereas the collection region 124of optical apparatus 100 should have a width that is smaller than thewidth of vessel 112, to prevent the inclusion of spectral informationfrom tissue outside of vessel 112. This is indicated in FIG. 2C whichshows multiple different types of blood vessels 112: a venule 112 a, anarteriole 112 b, an arterial capillary 112 c, and a venous capillary 112d located beneath the tissue surface 110, illuminated separately toallow collecting individual spectra corresponding to each blood vessel.PSF representations 132 are shown sized to illuminate the blood vesselsindividually, allowing spectral information to be exclusively collectedfrom each blood vessel for separate analysis. However, otherimplementations may be used in which the illumination and the detectionsystems can function on a group of vessels closely located havingsimilar physiological properties.

An imaging arrangement is provided, having sufficient resolution tolimit the collection of light only to that diffused from the vessel, andto enable visualization for selecting the blood vessel and spectralmeasurement of that reflected light. Referring back to FIG. 2A, theimaging channel comprising a beam splitter 126 coupled to a lens 128 toimage the focal plane of objective lens 108 onto a camera 130.Optionally, two crossed-polarizers 132 and 134 may be provided along theoptical path to reduce surface scattering and glare for reducing imagebackground and noise.

To improve accuracy and reduce measurement error, a reference spectrummay be measured at a location close to the target vessel, and used tocalibrate optical apparatus 100. Camera 130 may be used to identifyvessel 112 to allow maneuvering optical apparatus 100 to target theilluminating beam sequentially on vessel 112 or on a surroundingreference tissue sample for spectral analysis. For example, theapparatus or part of it, may be maneuvered to enable camera 130 tocollect either a reference spectrum from tissue surrounding vessel 112,or a spectrum exclusively from vessel 112. The collected spectra may beprovided to a processor 134 for analysis. Additionally or alternatively,one or more of the images captured by camera 130 may be provided toprocessor 134 for analysis accordingly, as described below in theimplementation of FIGS. 5A-5G. Processor 134 may render any of theimages, spectral information, and/or determined absorption levels on adisplay 136.

Referring to FIG. 3A-C, typical images of capillaries on the lip of asubject are shown, where the vessels appear as dark lines on a brightbackground due to the high absorption of blood compared to thesurrounding tissue. White stars 302, 304, and 306 indicate locationsinside vessel 112, and black stars 308, 310, and 312 indicate locationsoutside vessel 112. Spectra corresponding to location pairs (300, 306),(308, 302), and (304, 310) may be recorded and provided to processor134, to determine absorption levels using any of the methods describedbelow.

Referring to FIG. 4, a flowchart is shown of one method for measuringblood oxygenation levels. The optical apparatus is positioned at thesurface of a bodily tissue (Step 400), such that the region of interestis illuminated. The vessels lying under the bodily tissue are imagedusing the camera (Step 402), and a target vessel is selected (Step 404),and the focus is adjusted to obtain a sharp image of the target vessel(Step 406). The device is now maneuvered to illuminate surroundingtissue near, but not including the target vessel, and a referencespectrum emitted by the surrounding tissue is recorded by thespectrometer (Step 408). The optical apparatus is now maneuvered toorient the illumination onto the target vessel itself in both thelateral and axial (focus) dimensions (Step 410), and the spectrumemitted from the target vessel is recorded by the spectrometer (Step412). The blood oxygenation level corresponding to the target vessel isdetermined by analyzing the recorded target vessel spectrum as comparedwith the recorded surrounding tissue spectrum (Step 414). Steps 410-414may be repeated for additional blood vessels in proximity to thesurrounding tissue corresponding to the reference spectrum.

In some implementations, light source 102 may produce broadband light,or alternatively, light comprising several wavelength bands. As statedin the Background Section, conventional pulse oximetry generally useswavelengths at approximately 800 nm for the reference measurement, and asecond band, typically in the region of 950 nm, for determining theoxyhemoglobin level. The latter wavelength range is used forconventional oximetry measurements because that is a spectral regionhaving a large difference between the deoxy-hemoglobin absorbance andthe oxy-hemoglobin absorbance. In that spectral region, the light isweakly scattered by tissue and fairly weakly absorbed, but that the lowabsorption is not necessarily problematic for conventional oximetrymeasurements since measurements are taken over a comparatively largetissue and blood volume, typically of several millimeters cross section,such that there is a satisfactory transmitted signal. However, using themethods and systems of the present disclosure for imaging smallindividual blood vessels, which could be only tens of microns indiameter, the diffusely reflected optical intensity in that spectralregion is very small because of the low absorption in the vessel, makingit difficult to detect the diffusively reflected spectra in such a lowoptical attenuation medium. Furthermore, since the measurements are madeon blood vessels near the surface of the subject's tissue, it isimportant that the incident light does not penetrate too deeply into thetissue without attenuation, so that the absorption in the imaged bloodvessels can be successfully measured. Therefore, in the presentlydescribed system, it is more advisable to use a spectral region wherethe optical absorbance and scattering is higher, provided that thedifference in absorbance between the oxy- and the deoxy-hemoglobin issufficient to enable differentiation between them. Consequently, for thedifferentiating measurement, a detection wavelength in the 500 to 600nanometer range is used, where the absorbance is greater by almost twoorders of magnitude than in the near infra-red 700 to 900 nm region.Although the differential absorbance in the 500 to 600 nm region issmaller than in the infra-red, the greatly increased overall absorptionlevel enables a better signal-to-noise ratio to be achieved for smallsize measurements of individual blood vessels. Spectrometer 114 mayoptionally be replaced by a simpler wavelength discriminationarrangement, such as a combination of a dichroic mirror or filters, withone or more detectors, to measure several predefined spectral bands.Oxygenation levels may be measured by fitting the measured spectrum to alinear combination of the absorption curves for oxy- anddeoxy-hemoglobin, or alternatively by measuring the relative absorptionsof different spectral bands, as in pulse oximetry. The volume of theblood vessels from which the spectra are measured may be estimated fromthe image of the camera. Consequently, the average optical path lengthof the diffusively scattered light within the vessel may be estimated.Using these parameters, the total hemoglobin levels within the vesselmay be computed by adding the computed levels of deoxyhemoglobin andoxyhemoglobin. The level of carbon monoxide may also be measured usingthis approach by measuring the spectral characteristics ofcarboxyhemoglobin.

In some implementations, off-axis illumination may be used to providedark field illumination, to improve image quality of the imagingchannel, and resulting in less glare from the tissue surface andenhanced image contrast. Optionally, the widefield-imaging wavelengthillumination beam may be separated from the measurement beam usingdichroic mirrors.

Although the above described systems and methods use a large areaillumination beam and a small area imaging field of view, it is alsopossible for the optical setup of the spectral measurement to bereversed by providing a single-mode fiber for illuminating a relativelysmall spot on the tissue, and a relatively larger-core multimode fiberfor collecting the scattered light, resulting is the same overall systemPSF as shown in FIG. 2B while providing a higher illumination intensity.However, care must then be taken to ensure that the high incidentintensity does not cause tissue damage, and a larger illumination spotmay be advantageous since it allows taking measurements using higheroptical power without damaging the tissue.

Optionally, the optical fibers used for the illumination and collectionchannels may be replaced by a small-area light source and/or detector,with spatial filtering, in a free-space optical setup (not shown).

Reference is now made to FIGS. 5A to 5G, which illustrate an alternativemethod of determining the total hemoglobin and the oxygenation levelswithin blood vessels, only using the imaging functions of the apparatusof FIG. 2A, and without the need for focusing the incident light or theneed for spectrometric measurements. Measurements are taken at theisosbestic points and at wavelengths close to it. In this method, thedifference in reflectance (or absorbance) between the vessel and itsnearby tissue is determined by performing image processing on images ofthe blood vessel, and using the relative intensities of the lightdiffusively reflected from the blood vessel and its surrounding tissues,in order to extract information relating to the illumination absorption.These measurements assume that the light absorbance in the tissuesurrounding the vessel does not change appreciably with small changes inwavelength around the isosbestic point, and that the surrounding tissuecan therefore be used as a normalizing factor for compensating forrandom changes in the brightness or contrast of the images captured atthe isosbestic point and its neighboring measurement points. By thismeans, the intensities of the optical signals diffusely reflected fromthe blood vessel can be normalized, so that changes in the image qualityor brightness can be eliminated. The isosbestic wavelength measurement(FIG. 5C) allows measuring the total hemoglobin concentration. Byperforming these measurements at different wavelengths, the character ofthe oxygenation of the blood in the vessel can be obtained. Thewavelengths chosen should be an isosbestic wavelength of the oxy- anddeoxy-hemoglobin, such as that at 800 nm, or more preferably, asexplained hereinabove, the isosbestic wavelength in the green region, at570 nm, and two wavelengths, preferably on either side of the isosbesticpoint.

To illustrate this procedure, reference is now made to FIG. 5A, whichshows a graph comparing absorption spectra for hemoglobin 500 andoxyhemoglobin 502, indicating an isosbestic wavelength, λ_(isosbestic)and two wavelengths either side of the isosbestic wavelength. FIGS. 5B-Dshow three widefield images captured by camera 130 under illumination ofdifferent wavelengths. FIG. 5C shows a widefield image captured usingthe isosbestic wavelength λ_(isosbestic) indicated in FIG. 5A, FIG. 5Bshows a widefield image captured using a wavelength belowλ_(isosbestic), and FIG. 5D shows a widefield image captured using awavelength above λ_(isosbestic). The dashed lines across each imagerepresent an imaging line along which the intensity profile of thevessel may be measured. Although images 5B-D differ in both brightnessand contrast, they still indicate the absorption of the blood in thesmall vessel relative to its surrounding, as explained above. Thus,blood oxygenation of the blood vessel may be estimated by comparingFIGS. 5B-D, and the total hemoglobin concentration may be measured usingFIG. 5C corresponding to λ_(isosbestic). FIGS. 5E-G show absorptionspectra for the blood vessel, corresponding varying illuminationindicated for FIGS. 5B-D, respectively, where the vertical axis,indicated by I represents light intensity, and the horizontal axis,indicated by x represents the coordinate along the dashed lines in FIGS.5B-D. As is evident from FIGS. 5B-D, the peak absorption levels shiftwhen illuminated at wavelengths above, below and equal toλ_(isosbestic), with the relative change ΔI in the imaged intensity ofthe vessel indicating the optical absorbance through the vessel. Fromthe differences in ΔI at the three wavelengths measured, the ratio ofthe oxy- to deoxy-hemoglobin in the vessel can be obtained. Theadvantage of this method is that no spectrometric measurements need tobe made on the images, the sequential illumination by the threewavelengths being sufficient to determine the oxygenation level and thetotal hemoglobin simply from the image processing of the separate vesselimages captured by a simple camera.

The method of estimating the oxygenation and the total hemoglobin levelis now explained. According to the modified Beer-Lambert law, lightextinction after transmission through a region containing a vessel andthe intervening tissue is given by:

$\begin{matrix}{{A_{{vess}el}(\lambda)} = {{\left\lbrack {{c_{Hb}{ɛ_{Hb}(\lambda)}} + {c_{HbO_{2}}{ɛ_{HbO_{2}}(\lambda)}}} \right\rbrack \cdot p_{vessel}} + G_{tissue}}} & (1)\end{matrix}$

where A denotes the light extinction,

ε is the molar extinction coefficient,

c is the substance molar concentration,

p_(vessel) is the average optical path within the vessel, and

G is the signal loss due to scattering by the surrounding tissue.

Measuring light extinction at the tissue near the vessel, where there isno substantial absorption by blood absorption, only scattering by thetissue to a first approximation, and subtracting from the measurement ofthe extinction arising from the vessel yields:

$\begin{matrix}{{{A_{{vess}el}(\lambda)} - {A_{tissue}(\lambda)}} = {\left\lbrack {{c_{Hb}{ɛ_{Hb}(\lambda)}} + {c_{HbO_{2}}{ɛ_{HbO_{2}}(\lambda)}}} \right\rbrack \cdot p_{vessel}}} & (2)\end{matrix}$

Without knowledge of p_(vessel), only the relative concentration c_(HbO)₂ /(c_(HbO) ₂ +c_(Hb)) can be calculated, providing the bloodoxidization parameter. However, when p_(vessel) is known, either bymeasurement or by estimation, it is possible to fit the measuredabsorption spectra using both concentration parameters and to obtain thetotal hemoglobin concentration:

$\begin{matrix}{c_{tot} = {c_{Hb} + c_{HbO_{2}}}} & (3)\end{matrix}$

Using widefield images for hemoglobin measurement, the extinctiondifference for the isosbestic image is given by:

$\begin{matrix}{{\left\lbrack {A_{vessel} - A_{tissue}} \right\rbrack\left( \lambda_{iso} \right)} = {c_{tot}p_{vessel}{ɛ_{HbO_{2}}\left( \lambda_{iso} \right)}}} & (4)\end{matrix}$

since ε_(Hb)=ε_(HbO) ₂ at the isosbestic wavelength. Therefore, ifp_(vessel) is known, either by calculation, or independent measurement,c_(tot) may be calculated directly from Eq. (4) by comparing lightextinctions in and nearby the vessel.

Medium sized vessels may be assumed to have a circular cross section,leading these vessels to appear darker at their center and brighter atthe margins due to either the vessel having greater optical thickness atits center or alternatively, due to the higher concentration of bloodflow at the center of the vessel. By assuming that the vessel has anapproximately circular cross section, the true optical path lengthtraveled by the light reaching the camera or the single detector can becomputed. The true path-length is equal to the vessel diameter times theDPF (differential path-length factor). If there is no scattering, DPF=1.For large vessels, DPF can equal 2 or even more. The DPF can bepredicted for different vessel sizes using numerical simulation or MonteCarlo light scattering simulations, or by measuring these effects ontissue models The p_(vessel) may thus be estimated or indirectlymeasured for every lateral location along the dashed lines in FIGS.5B-D, corresponding to the peaks, valleys, and points of intersection ofcurves 500 and 502, to obtain p_(vessel)(x). The total hemoglobinconcentration c_(tot) may then be estimated using a simple fit:

$\begin{matrix}{{{A_{{vess}el}(x)} - A_{tissue}} = {c_{tot}ɛ_{HbO_{2}}{p_{vessel}(x)}}} & (5)\end{matrix}$

If the camera image is of high quality, the transverse profile of thedark vessel along the dashed lines in FIGS. 5B-5D, can assist inimproving the accuracy, by using the extra data points along the vesselcross section, i.e. the darker center and brighter edges. Using theappropriate simulations, the single DPF value can then be replaced by amore complex analysis of the light emerging from the vessel. In thiscase, this is no longer a single path-length value and a singleabsorption measurement, but instead a more complex 2D/3D modeling of thevessel, combined with fitting the results to the 2D camera image.

The above described method may be performed on even small capillaries,provided that the camera has sufficient resolution. However there is aproblem with very small capillaries, because of the nature of the sparesblood flow through them, which may result in intermittent detection ofclusters of red blood cells and their concomitant hemoglobin content. Onthe other hand, if the vessel selected is too large, the illuminatinglight may not penetrate through its entire depth, and provide anaccurate reflection image of the conditions through the entire bloodvessel. In order to overcome these two extreme situations, it ispossible to select an area containing multiple vessels, of a sizesufficiently small not to cause inaccuracy problems in the generation ofthe reflection image, and to perform the measurements sequentially onone vessel after the other, and to integrate the results to obtain anaccurate measure of the total hemoglobin concentration.

One of the advantages of the above described methods of measuring thetotal hemoglobin level, is the comparative simplicity of the apparatusneeded for such measurements. The apparatus need use only a fewcomponents and functions of the system shown in FIG. 2A. Theillumination can be supplied by any suitable external source 102, andcan be applied to the sample tissue by wide-field illumination infree-space. Instead of the spectrometer 114, a simple wavelengthselection arrangement, such as a dichroic mirror or filter may be usedto enable the optical measurements to be performed only at theisosbestic wavelength point, or close to it. The location and lightdetection of the region to be measured can be accomplished using acamera with resolution sufficient to image the blood vessels with thedetail required to perform the above described image processingprocedures. That camera may also be used in providing the imagesnecessary for determining the optical path depth through the bloodvessel. The camera may be equipped with a narrow-field confocaldetection system to enable high resolution selection of the region to beimaged. The processor is adapted to perform the image analysis andcompute the total hemoglobin content of the blood vessel, using themethods suggested by equations (1) to (5), and the calculation methodsdescribed in the previous two paragraphs of this disclosure. The systemitself therefore becomes very simple in construction.

The above derivation has been described for a single isosbesticwavelength, but the optical measurement may also be obtained by usingthe previously described technique of multiple wavelengths and spectralanalysis, thereby providing increased accuracy and the added informationof an oxygenation determination. Additionally, although the full fieldexamination and imaging provides a particularly simple method ofperforming the optical measurement, it is also possible to use aconfocal method using a single point or small region measurement of theextinction at the blood vessel, together with a single point or smallregion reference measurement and the surrounding tissue.

Furthermore, the most accurate and the simplest method of implementingthe above described methods and systems, are to perform the basicimaging of the blood vessel and its surrounding tissue at an isosbesticwavelength of oxyhemoglobin and deoxyhemoglobin, such that the effect ofthe oxygenation level of the blood is eliminated.

This results in the simplest processing of the data, and the mostaccurate results. However it is also possible to operate the describedmethods and systems at a wavelength in the region of the isosbesticwavelength, where the difference in extinction coefficients ofoxyhemoglobin and deoxyhemoglobin may be minimal, and the presentdisclosure is intended to cover such cases also. Furthermore, insituations where the oxygenation level of the patient is known, such asby use of a simple pulse oximetry measurement, since the spectralabsorptions of oxyhemoglobin and deoxyhemoglobin are well known, it ispossible to operate at a wavelength distant from an isosbestic point,and to use in the calculations based on the imaged intensities of theblood vessel and its surroundings, the extinction coefficients at thatwavelength for the measured oxygenation level, and thus to derive thetotal hemoglobin level. As an even further method, it is possible tomake an approximate estimate of the oxygenation level of the subject,based possibly on the clinical state of the subject, and to use thatestimate in the derivation of the total hemoglobin calculation at anywavelength other than an isosbestic wavelength. For instance, it isknown that in reasonably healthy subjects, an approximate level ofoxygenation is of the order of 90% for an artery and 60% for a vein.However it is clear that any of these methods are likely to involve lessaccuracy than the previously described method using a measurement at orvery close to an isosbestic wavelength.

The descriptions of the various embodiments of the present inventionhave been presented for purposes of illustration, but are not intendedto be exhaustive or limited to the embodiments disclosed. Manymodifications and variations will be apparent to those of ordinary skillin the art without departing from the scope and spirit of the describedembodiments. The terminology used herein was chosen to best explain theprinciples of the embodiments, the practical application or technicalimprovement over technologies found in the marketplace, or to enableothers of ordinary skill in the art to understand the embodimentsdisclosed herein.

What is claimed is:
 1. A method for measuring total hemoglobinconcentration in a blood vessel, comprising: illuminating a bodilytissue containing the blood vessel; generating images of the bloodvessel and its surrounding tissue at a first wavelength in the spectralregion of an isosbestic wavelength of oxyhemoglobin anddeoxy-hemoglobin; determining at that first wavelength a region ofinterest containing image brightness of the blood vessel and of thesurrounding tissue; estimating at least one geometrical property of thevessel; and using the at least one estimated geometrical property andthe image containing the blood vessel and the surrounding tissue todetermine the total hemoglobin concentration in the blood flowing in theblood vessel.
 2. The method according to claim 1, wherein the at leastone geometrical property is selected from a list consisting of: a sizeof the vessel, a length of the vessel, a cross section of the vessel, adepth of the vessel, a circularity of the vessel, and the vessel'sdiameter.
 3. The method according to claim 1, wherein the totalhemoglobin is determined using comparison between the generated imagesand images generated using numerical simulation based on the estimatedgeometrical property of the vessel.
 4. The method according to claim 1,further comprising: generating images of the blood vessel and itssurrounding tissue at at least one additional wavelength in proximity tothe first wavelength; determining the comparative imaged intensities ofthe blood vessel at the first wavelength and at the at least oneadditional wavelength in proximity thereto; and using the comparativeimaged intensities of the blood vessel to determine the oxygenabsorption level in the blood.
 5. The method of claim 3, wherein the atleast one additional wavelength is a range of multiple wavelengths.
 6. Amethod according to claim 1, wherein the images of the blood vessel andits surrounding tissue are obtained from a camera system.
 7. A methodaccording to claim 1, wherein the images of the blood vessel and itssurrounding tissue are obtained from a narrow field confocal microscopesystem.
 8. The method according to claim 1, wherein the first wavelengthimages are generated by illuminating at the first wavelength.
 9. Themethod according to claim 1, wherein the first wavelength images aregenerated by imaging at the first wavelength.
 10. A system for measuringtotal hemoglobin concentration in a blood vessel, comprising: a lightsource configured to illuminate the blood vessel and its surroundingtissue; a camera configured to generate images of the blood vessel andits surrounding tissue at a first wavelength in the spectral region ofan isosbestic wavelength of oxyhemoglobin and deoxy-hemoglobin; and aprocessor configured to: (i) determine from at least one of the imagesgenerated at the first wavelength, a region of interest containing imagebrightness of the blood vessel and of the surrounding tissue; (ii)estimate at least one geometrical property of the vessel; and (iii) usethe at least one estimated geometrical property of the vessel and theimaged containing the blood vessel and of the surrounding tissue todetermine the total hemoglobin concentration in the blood flowing in theblood vessel.
 11. The system of claim 10, wherein the at least onegeometrical property is selected from a list consisting of: a size ofthe vessel, a length of the vessel, a cross section of the vessel, adepth of the vessel, a circularity of the vessel, and the vessel'sdiameter.
 12. A system according to claim 10, wherein the camera isconfigured to generate images of the blood vessel and its surroundingtissue at at least one additional wavelength in proximity to the firstwavelength; and wherein the processor is further configured to (iv)determine the comparative imaged intensities of the blood vessel at thefirst wavelength and at the at least one additional wavelength inproximity thereto; and (v) use the comparative imaged intensities of theblood vessel to determine the oxygen absorption level in the blood. 13.The system of claim 12, wherein the at least one additional wavelengthis a range of multiple wavelengths.
 14. A system according to claim 10,wherein the light source is configured to provide wide fieldillumination.
 15. A system according to claim 10, wherein the cameracomprises a confocal microscope system configured to image the bloodvessel and its surrounding tissue.
 16. A system according to claim 10,wherein the first wavelength images are generated by illuminating at thefirst wavelength.
 17. A system according to claim 10, wherein the firstwavelength images are generated by a light source providing illuminationat the first wavelength.
 18. A system according to claim 10, wherein thefirst wavelength images are generated by at least one filter disposed inthe optical path of the illumination either before incidence on theblood vessel and its surrounding tissue, or after reflection from theblood vessel and its surrounding tissue.